PGD and PGS: The Use of FISH, CGH and aCGH for Identifying Chromosome Mosaicism Correlation

INTRODUCTION

Preimplantation Genetic Diagnosis and Preimplantation Genetic Screening (PGD/PGS) for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying non-affected and/or euploid embryos in a cohort produced during an IVF treatment cycle. The main goal of Preimplantation Genetic Diagnosis and Preimplantation Genetic Screening (PGD/PGS), which is the biopsy of one or more cells from a preimplantation embryo followed by the ploidy analysis of these cells and finally transfer of those embryos deemed to be euploid, is to define whether an embryo is affected by a monogenic disease and/or chromosomal impairments, thus preventing the implantation of a symptomatic fetus and/or limiting the risks underlying the transfer of chromosomally abnormal embryos (mainly implantation failures and miscarriages). In synthesis, PGD/PGS is a powerful tool to reach the goal of a pregnancy and attenuate its adverse events. In order to achieve this goal, it is mandatory not to significantly harm the embryo during the biopsy and to preserve its viability and reproductive potential. First do not harm is a dogma in clinical practice that perfectly applies also to this context.

The weak correlation between conventional methods of embryo evaluation and chromosomal complement has led to the introduction of preimplantation genetic screening (PGS) in in vitro fertilization (IVF) cycles as a complementary tool aiming at selecting euploid embryos and improving delivery rate per transfer [Munné et al., 1993]. Chromosomal abnormalities are responsible for a high proportion of embryonic loss, as already postulated by data from early abortions [Plachot et al., 1988; Burgoyne et al., 1991]. Preimplantation genetic diagnosis (PGD) offers an alternative for women to carry a fetus unaffected by hereditary diseases. The value of the strategy of blastocyst biopsy, cryopreservation [Recently, vitrification has been verified to be efficient for cryopreservation of blastocysts [Liebermann, 2009]. Cryopreservation of biopsied blastocysts would permit appropriate time for genetic diagnosis. Furthermore, cryopreservation of biopsied blastocysts may avoid late ovarian hyperstimulation syndrome (OHSS) as hCG produced from implanted embryos may induce late and prolonged OHSS after fresh transfer in the high ovarian responders [Chang et al., 2011; Chen et al., 2011], and thawed embryo transfer for preimplantation genetic diagnosis (PGD) of monogenic diseases remains indefinable and deserves further investigation.

Aneuploidy is the principal genetic factor affecting reproductive success. A great number of morphologically normal embryos either do not implant or spontaneously abort early in pregnancy because their chromosome number deviates from the normal diploid 46. Numerous studies have examined the chromosomes of gametes and preimplantation embryos, demonstrating that aneuploidy can arise during meiosis or after fertilization. Most meiotic errors are derived from the oocyte. Investigations using a wide range of cytogenetic techniques have confirmed the high prevalence of chromosome anomalies of maternal origin and shown that aneuploidy increases in frequency with advancing female age [Kuliev et al., 2003; Pellestor et al., 2003; Fragouli et al., 2006, 2010; Fishel et al., 2010].

Chromosomal abnormalities arising after fertilization, at the cleavage stage, have also been the subject of cytogenetic study. The results obtained suggest that most anomalies occur during the first three mitotic divisions, leading to chromosomal mosaicism, the presence of two or more karyotypically distinct cell lines within the same embryo [Delhanty et al., 1997; Munné et al., 2002; Katz–Jaffe et al., 2004]. During in vitro fertilization (IVF) cycles, the embryonic cohort is asynchronous in development and trophectoderm (TE) biopsy can equally be performed on Day 5, Day 6 or even Day 7 post–fertilization and on blastocysts of different morphological quality. It is still unknown whether blastocyst morphology and developmental rate relates to the embryo chromosomal constitution. Only one study that attempted to correlate embryo morphology and aneuploidy as determined by comprehensive chromosome screening (CCS) on trophectoderm (TE) biopsies [Alfarawati et al., 2011] showed a weak correlation between aneuploidy and blastocyst morphology. However, the biopsy procedures used in that study used zona opening at the cleavage stage of embryo development to promote trophectoderm (TE) cell herniation and facilitate the blastocyst biopsy procedure. This method may have introduced interference in embryo development from the cleavage to the blastocyst stage, lowering the reliability of the study when the data are extended to the general population of blastocysts obtained during regular IVF cycles.

Furthermore, no studies have attempted to correlate conventional parameters of blastocyst evaluation with euploid embryo viability in frozen embryo transfer (FET) cycles. It is still unknown whether euploid blastocysts with a different morphology and developmental rate implant at a different rate. This knowledge may be useful to further enhance the selection among euploid embryos.

Variable morphology associated with different rates of cleavage and degrees of fragmentation are characteristic of preimplantation embryos [Trounson, 1983; Bolton et al., 1989; Dorkras et al, 1993; Hardy, 1994; Alikani et al., 1999]. However, it is not clear whether this is due to fertilization and culture in vitro, to follicular stimulation with high doses of gonadotrophins, or if it is a specific embryo characteristic. In any case, the detrimental effects associated with fragmentation, and delayed or arrested cleavage, could substantially contribute to the reduced viability that characterizes the embryo. This hypothesis has been confirmed by recent studies which report a strong correlation between slow cleavage rate of embryos and their chromosomal normality [Munné et al., 1995; Magli et al., 1998]. Therefore, it has been recommended that culturing embryos to the blastocyst stage instead of early cleavage stages will enable the selection and identification of healthy, chromosomally normal embryos endowed with a high potential for implantation [Janny and Ménézo, 1996; Jones et al., 1998a]. If this is true, a mechanism of natural selection may operate during preimplantation development which eliminates abnormal embryos or selectively allocates aneuploid cells to the trophectoderm (TE) and euploid cells to the inner cell mass (ICM) [Hardy et al., 1989]. This mechanism could become active at the time when a clear polarity arises in the embryo. It has recently been reported [Evsikov and Verlinsky 1998] that, after fluorescent in–situ hybridization (FISH) analysis for the chromosomes 13, 18 and 21 of blastocysts, mosaicism is present in the inner cell mass (ICM). This observation would not support the hypothesis of a selective or preferential allocation of euploid cells in the inner cell mass (ICM).

The prevalence of aneuploidy in embryos provides a likely explanation for the relatively low success and the high abortion rate observed during assisted reproductive treatment cycles [Spandorfer et al., 2004; Menasha et al., 2005]. It is believed that, in most cases, aneuploidy causes embryos to either fail to implant after transfer or spontaneously abort early in gestation [Macklon et al., 2002]. It has been shown that normal preimplantation embryo development does not correlate with euploidy. Both blastocysts of good and poor morphology have almost the same probability of carrying chromosomal abnormalities [Fragouli et al., 2008; Alfarawati et al., 2011]. This poor correlation of conventional embryo selection methods and chromosomal complement led to the introduction of preimplantation genetic screening (PGS) as a complementary treatment during IVF cycles to avoid the transfer of aneuploid embryos. It was thought that implementing preimplantation genetic screening (PGS) would improve ‘top–quality’ embryo selection process. Two main reasons have been put forward to explain this: (I) the cytogenetic investigation methods used, such as fluorescence in situ hybridization (FISH), are able to detect only copy number variation of a few chromosomes and have a considerable per chromosome diagnostic error rate when applied on single cells [Munné et al., 2002; Baart et al., 2004; Li et al., 2005; Colls et al., 2007; Magli et al., 2007; Harper and Harton, 2010, b; Gutiérrez-Mateo et al., 2011]; (II) the stage of analysis and the cell type used to infer the chromosome copy number of the embryo do not always correlate with the embryo as a whole [Vanneste et al., 2009a].

In the preimplantation genetic screening (PGS) field there is an ongoing debate about the optimal biopsy stage for preimplantation genetic screening (PGS). This is a result of the lack of understanding of how aneuploidy arises in the embryo. To date, most of the cytogenetic data obtained during preimplantation genetic screening (PGS) investigations have been derived through the analysis of cells at isolated points in the preimplantation window, thus potentially missing critical information on chromosomal segregation. Understanding the chromosome segregation patterns during preimplantation development holds the potential to significantly increase the success rates of IVF. To date, most analyses of cleavage–stage embryos have involved testing of small numbers of chromosomes using fluorescence in situ hybridization (FISH). While this approach has provided a useful insight into aneuploidy during early embryo development, most chromosomes remain untested and the cytogenetic assessment is therefore incomplete. Rare studies involving comprehensive chromosomal analysis, using comparative genomic hybridization (CGH), have suggested that ∼50% of embryos are aneuploid in every cell, with the remaining embryos equally divided into those containing only normal cells and those composed of a mixture of abnormal and normal cells [Voullaire et al., 2000; Wells and Delhanty, 2000].

There are three potential sources of embryonic genetic material for preimplantation genetic analysis: polar bodies (biopsy of the first polar body from oocytes before sperm insemination or biopsy of both polar bodies from oocytes after sperm insemination), blastomeres from cleavage stage embryos and trophectoderm cells from embryos at the blastocyst stage.

Testing at the polar body (PB) stage is the least accurate mainly due to the high incidence of post–zygotic events. This suggests that postponing the time of biopsy to the blastocyst stage of preimplantation embryo development may provide the most reliable results for preimplantation genetic screening (PGS). In clinical practice, polar body biopsy has been used primarily for aneuploidy screening [Verlinsky et al., 1996] and less commonly for the detection of maternal transmission of single gene defects [Verlinsky et al., 1997] such as β–thalassaemia [Kuliev et al., 1998]. According to the ESHRE preimplantation genetic screening (PGS) Consortium data collections, cleavage stage biopsy, usually involving aspiration of one to two blastomeres, is the most common approach for attaining embryonic genetic material for preimplantation genetic screening (PGS) analysis [Harper et al., 2006]. Embryo biopsy at the cleavage stage (6– to 8–cell stage) with retrieval of one or two blastomeres is the most common approach for preimplantation genetic diagnosis (PGD) of monogenic diseases [Harper et al., 2012]. However, the innate dilemma of cleavage–stage biopsy is that it obtains the very limited genetic material and time available for performing analysis. The risks of misdiagnosis may occur in polymerase chain reaction (PCR)–based protocols. Some misdiagnoses with preimplantation genetic diagnosis (PGD) have been reported in the literature [Wilton et al., 2009]. Precompaction eight–cell embryos are usually biopsied early on day 3 (insemination on day 0), and following genetic diagnosis, the embryo transfer may be performed on the same day [Boada et al., 1998] or delayed to day 4 or until the embryo has reached the blastocyst stage.

The second major issue for the outcome of preimplantation genetic screening (PGS) programs relates to the type of cell biopsied. The biopsied sample should be representative of the embryos’ chromosomal constitution and viability after transfer. There are different possible sources of genetic material for testing in the preimplantation period in patients undergoing an IVF cycle: (I) the first and second polar bodies (PBs) (PB approach); (II) one or two cells biopsied from 5– to 10–cell cleavage–stage embryos on Day 3 and (3) several trophoblast cells (usually 5–10) sampled from the blastocyst [Capalbo et al., 2013].

Each of these stages presents with specific diagnostic advantages as well as critical limitations that relate to aneuploidy genesis during both meiosis and the preimplantation period of embryo development [Angell, 1991; Hassold and Hunt, 2001; Delhanty, 2005; Vanneste et al., 2009b; Northrop et al., 2010; Handyside et al., 2012; van Echten–Arends et al., 2011]. In the preimplantation genetic screening (PGS) field there is an ongoing debate about the optimal stage for preimplantation genetic screening (PGS) as a consequence of the lack of a complete understanding of the genesis of embryo aneuploidy. Indeed, most of the cytogenetic data obtained during preimplantation genetic screening (PGS) investigations have been derived through the analysis of cells at isolated points in the preimplantation window, thus potentially missing critical previous or subsequent information on chromosomal segregation. Understanding the chromosome segregation patterns during preimplantation development holds the potential to significantly increase the success rates of in vitro fertilization (IVF) therapy cycles.

A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed by experts. Cleavage stage blastomere biopsy still represents the most commonly used method nowadays, although this approach has been shown to have a negative impact on embryo viability and implantation potential. Polar body biopsy has been proposed as an alternative to embryo biopsy especially for aneuploidy testing. However, to date no sufficiently powered study has clarified the impact of this procedure on embryo reproductive competence. Blastocyst stage biopsy represents nowadays the safest approach not to impact embryo implantation potential. For this reason, as well as for the evidences of a higher consistency of the molecular analysis when performed on trophectoderm cells, blastocyst biopsy implementation is gradually increasing worldwide.

The main disadvantage of preimplantation genetic diagnosis (PGD) based on analysis of polar body or blastomere biopsy procedures is the limited amount of material available for genetic analysis. When diagnosing monogenic disorders in single cells using PCR–based protocols, there is a high risk of PCR failure (no result) and allele dropout (ADO) (incomplete result), potentially resulting in a reduced number of unaffected embryos available for transfer. Increasing the amount of starting DNA template should in principle increase the sensitivity and reliability of genetic diagnosis. Consequently, the biopsy of multiple trophectoderm cells from the blastocyst rather than a single cell from cleavage stage embryos should potentially lead to improved preimplantation genetic diagnosis (PGD) outcome for patients. Blastocyst trophectoderm biopsy using micromanipulation methods was first reported by Dorkas et al. (1990), although not in the context of clinical application. The development of noncontact lasers has greatly facilitated trophectoderm biopsy, first to make a hole in the zona and secondly, following a period in culture, to excise trophectoderm cells [Veiga et al., 1997]. Pregnancies following blastocyst biopsy and preimplantation genetic diagnosis (PGD) have recently been reported [de Boer et al., 2004; Kokkali et al., 2005].

(2) PRE–BIOPSY LABORATORY PROCEDURES AND BLASTOCYST GRADING

Controlled ovarian stimulation, oocyte collection and denudation should be performed using a GnRH–agonist long protocol and oocyte collection should be performed at 35–36 hours post–hCG administration [Capalbo et al, 2013a; Ubaldi et al., 2010]. Denudation of the oocyte from the cumulus oophorus should be performed by a brief exposure to 40 IU/ml hyaluronidase solution in fertilization media, followed by mechanical removal of all the corona radiata with the use of plastic pipettes of defined diameters. The denudation procedure should be performed in a controlled (6% CO2 at 37°) environment between 37 hours and 40 hours post–hCG administration. Particular attention should be paid to the removal of all adhering cumulus and coronal cells with the aim of avoiding maternal DNA contamination during the amplification steps [Capalbo et al., 2013a].

At 16–18 hours post–ICSI, oocytes should be assessed for the presence of pronuclei. Those displaying two pronuclei should be sequentially cultured further in separate 35 µl microdrops (Sage) up to blastocyst stage (Day 5/ Day 6) in a humidified atmosphere containing 5% O2 and 6% CO2. Expanding and expanded blastocysts should undergo biopsy of TE cells and cryopreservation on Day 5. Cavitating morulas should be transferred to a fresh individual 35 µl drop of blastocyst medium with 15% Serum Protein Substitute and biopsy should be attempted 24 hours later on Day 6 or 48 hours later on Day 7 [Capalbo et al., 2014].

Blastocyst quality should be assessed immediately before TE biopsy, defined according to the criteria presented by Gardner and Schoolcraft (1999) and categorized in four groups: excellent, group 1 (≥3AA); good, group 2 (3,4,5,6, AB and BA); average, group 3 (3,4,5,6 BB, AC and CA); poor, group 4 (≤3BB) based on inner cell mass (ICM) and TE quality score [Capalbo et al., 2014]. Furthermore, individual inner cell mass (ICM) and TE scores should recorded. The TE should be assigned one of the following grades: A: many cells organized in epithelium; B: several cells organized in loose epithelium; or C: few large cells. The inner cell mass (ICM) should be assigned oneof the following grades: A: numerous tightly packed cells; B: several and loosely packed cells; or C: very few cells. All embryo grading should be reviewed in real–time by senior embryologist for verificationand consistency [Capalbo et al., 2014].

All the biopsy procedures were performed on the heated stage of a microscope, equipped with micromanipulation tools, in dishes prepared with three droplets of 10 μl of HEPES–buffered medium (Sage) overlaid with pre–equilibrated mineral oil. A diode laser was used to assist the opening of a 10–20 μm hole in the zona pellucida by 2–4 laser shots. At all the biopsy stages, an attempt was made to use the initial zona breach to extract the target cells. A PB biopsy was performed sequentially by aspiration with a PB aspiration pipette. The first polar body (PB1) was biopsied immediately before intracytoplasmic sperm injection (ICSI) and the second polar body (PB2) was biopsied 16–18 hours after intracytoplasmic sperm injection (ICSI), allowing the distinction between the polar bodies (PBs) in all cases and avoiding the degradation of the genetic material. Occasionally, a second hole was required for sampling the second polar body (PB). A cleavage–stage embryo biopsy was performed on Day 3 of embryo development by blastomere extrusion of one blastomere from embryos reaching at least the six–cell stage as described by Tarín and Handyside, 1993 [Tarín, Handyside 1993]. The cleavage–stage biopsy was performed in calcium–magnesium–free HEPES buffered medium (Sage) using a blastomere aspiration pipette (Research instruments). On Day 5 or 6 of embryo development expanding and expanded blastocysts with or without herniating cells underwent TE biopsy. Five to 10 TE cells were aspirated into the TE biopsy pipette (Research instruments) followed by laser–assisted removal of the target cells from the body of the embryo. All embryos on Day 5, which did not reach the expanding blastocyst stage, were transferred to fresh individual 35 mL drop of blastocyst medium (Sage) and a biopsy was performed on Day 6 if the appropriate stage of development was reached. aCGH was performed only on the material obtained from oocytes/embryos where at least further biopsy was performed on Day 3 [Capalbo et al., 2013].

Blastocyst biopsy procedure

On Day 5, Day 6 or Day 7 of embryo development expanding and expanded blastocysts with or without herniating cells underwent TE biopsy. However, the main difference with previous reports describing a blastocyst biopsy procedure [Schoolcraft et al., 2010; Alfarawati et al., 2011] is that opening of the zona pellucida at the cleavage stage of embryo development is not necessarily being used in blastocyst stage biopsy program.

In the six– to eight–cell cleavage stage biopsy group, embryos were selected for biopsy based on morphological criteria [Rijnders and Jansen, 1998]. However, embryo morphology on day 3 does not predict developmental potential nor does it evaluate the numerical chromosomal status [Magli et al., 2000; Staessen et al., 2004]. Indeed, in our study, 22.9% of the cleavage stage embryos were unsuitable for biopsy on day 3 (<6 cells). Blastocyst biopsy provides a means for selecting embryos for biopsy that have at least demonstrated the potential of continued development under embryonic genomic control. Thus, selection is based on more objective criteria. In addition, embryos selected for biopsy on day 5 carry a lower risk of being aneuploid [Magli et al., 2000; Staessen et al., 2004], and although development to the blastocyst stage is not a guarantee of chromosomal normality, the majority of embryos that fail to continue in extended culture show multiple aneuploidies for chromosomes X, Y, 16, 18 and 21 [Magli et al., 2000]. In addition, observations suggest that the level of mosaicism in the trophectoderm is not higher than that seen in the inner cell mass of the blastocyst [Magli et al., 2000; Evsikov and Verlinsky, 1998]. The occurrence of mosaicism in blastocysts (either generalized mosaicism or a chromosomal dichotomy between inner cell mass and trophectoderm) could have consequences on the accuracy of PGD, but for monogenic disorders diagnosed with PCR–based methods, it is unlikely to lead to a misdiagnosis resulting in transfer of an affected embryo.

Array CGH analysis and embryo classification

All TE biopsies should be washed in sterile phosphate–buffered saline (PBS) solution in a laminar flow cabinet to avoid any contamination of the sample, placed in microcentrifuge tubes containing 2 µl PBS and then processed for array comparative genome hybridization (aCGH) analysis according to the 24–sure protocol [Capalbo et al., 2014]. Visualization and reporting of aneuploidy should be performed using the Bluefuse Software on a per chromosome basis as previously described [Capalbo et al., 2013a]. The copy number and segmental calls should be based on the deviation from the acceptable thresholds, as follows: monosomy of −0.48 and lower, euploid 0 and trisomy +0.38 and higher. This is based on a standard log2 ratio with X–separation of +0.48. Positive (5–cell samples of normal male fibroblast cell lines) and negative controls (empty biopsy medium) should be included in every run. To investigate whether different degrees of chromosomal errors may result in a different developmental behavior of the embryos, the aneuploid aCGH results should be further separated as single or double aneuploid, and as complex aneuploid when more than two chromosome errors were observed in the TE cell samples [Capalbo et al., 2014].

Blastocyst morphology and aneuploidy screening data

Among the embryological variables of blastocyst evaluation assessed (embryo quality, day of biopsy, and inner cell mass (ICM and TE scores) the logistic regression analysis adjusted for the IVF center and female age showed that only blastocyst morphology is predictive of the comprehensive chromosome screening (CCS) data [Capalbo et al., 2014].

Embryos with the highest morphological scores show a higher euploidy rate compared with lower quality embryos. In accordance with a proposed rate, the euploidy can be rated as 56.4, 39.1, 42.8 and 25.5% in the excellent, good, average and poor blastocyst morphology groups, respectively. A diagnosis of complex aneuploidy can be also associated with blastocyst morphology (P < 0.01), as it was practically performed, with 6.8, 15.2, 17.4 and 27.5% of excellent, good, average and poor–quality embryos showing multiple chromosome errors. ICM and TE scores were also independently associated with aneuploidy screening data [Capalbo et al., 2014].

A straightforward point for the purpose of Capalbo A., et al. (2014) study relied on the strategy of blastocyst biopsy used during the Preimplantation Genetic Screening (PGS) cycles. Contrary to previous approaches proposed for blastocyst biopsy [Schoolcraft et al., 2010; Alfarawati et al., 2011], the scientists recommend that no laser–assisted breach in the zona pellucida should be performed and a conventional embryo culture system up to the expanded blastocyst stage should be conducted [Capalbo et al., et al. 2014]. It is emphasized that this aspect helps to avoid any interference on blastocyst development as well as associated stress due to warming during the laser shooting at the cleavage stage. Furthermore, almost all embryos should be biopsied at the same expanded stage of development, with morphology and day of biopsy post fertilization being the only differences among the blastocysts analyzed. All these clues together provide a more representative picture of the relationship between conventional blastocyst evaluation, comprehensive chromosome screening (CCS) data and viability [Capalbo et al., 2014].

To confirm the moderate association between aneuploidy and several distinct features of blastocyst morphology reported in an earlier study [Alfarawati et al., 2011], it was performed, analyzed and distinguished that increased aneuploidy rate among blastocysts with poor morphologic scores and a greater likelihood of euploidy for embryos with good scores can be observed. However, this association is weak with a significant proportion of aneuploid embryos capable of achieving the highest morphologic scores (52% of excellent and good quality blastocysts). Accordingly, traditional morphology–based selection could not be relied on to significantly increase the likelihood of transferring chromosomally normal embryos in the absence of Preimplantation Genetic Screening (PGS) [Capalbo et al., 2014].

Another embryological parameter analyzed in Capalbo et al. (2014) study was the timing of embryo development to the blastocyst stage. Embryos reaching the expanded blastocyst stage on Day 6 showed a similar risk of being aneuploid as faster growing ones. Furthermore, even if based on a small sample size, Day 7 blastocysts present the same aneuploidy rate. This evidence suggests that the timing of blastocyst formation is not linked to or affected by chromosomal abnormalities, showing that delayed blastulation was not associated with increased aneuploidy rates [Alfarawati et al., 2011; Kroener et al., 2012; Capalbo et al., 2014].

Additionally, the examination of the frozen embryo transfer (FET) outcomes of blastocysts only provided a powerful study model to investigate the predictive role of conventional embryological evaluation when the prevailing confounding factor of chromosomal abnormalities is excluded from the analysis. Interestingly, in this data analysis blastocyst morphology and developmental rate were not associated with embryo viability. Lower-quality euploid embryos yielded the same ongoing implantation rate (45.2%) compared with blastocysts evaluated as of excellent and good morphological quality (51.4%) [Capalbo et al., 2014]. These findings also agree well with a recent study showing that excellent clinical outcomes are obtained when comprehensive chromosome screening based (CCS–based) selection was different than morphology–based selection [Forman et al., 2013]. Thus, morphology seems not to be an additional parameter to consider when multiple euploid embryos are available for transfer [Capalbo et al., 2014]. As a consequence, the recognized association between conventional evaluation of blastocyst morphology and embryo viability [Heitmann et al., 2013] can be mainly ascribed to the observed relationship between morphology and aneuploidy [Capalbo et al., 2014].

The association between blastocyst morphology and aneuploidy explains the higher implantation potential of good quality embryos reported during conventional IVF cycles. The relationship between morphology and aneuploidy screening data suggests that when Preimplantation Genetic Screening (PGS) is not available, blastocyst morphology should be used to slightly reduce the risk of transferring aneuploid embryos. However, traditional morphology–based selection cannot be used as an alternative to Preimplantation Genetic Screening (PGS) to minimize the risk of transferring chromosomally abnormal embryos. In addition, the commonly used parameters of blastocyst evaluation are not good indicators to improve the selection among euploid embryos. Thus, provided that the expanded stage is reached, all poor morphology and slower growing embryos have to be biopsied and similarly considered for frozen embryo transfer (FET) cycles. This knowledge will be of critical importance to achieve similar cumulative live birth rates in Preimplantation Genetic Screening (PGS) programs compared with conventional in vitro fertilization (IVF) avoiding the potential for exclusion of low quality but viable embryos from the biopsy and transfer procedures. It is expected that the more the genetic and molecular features of embryo development are characterized, the more the role of traditional morphology–based selection will be replaced in in vitro fertilization (IVF). Future research to identify non–invasive biomarkers of reproductive potential may further enhance selection among euploid blastocysts [Capalbo et al., 2014].

In addition, a new clinical protocol for TE biopsy suitable for both hatching and non-hatching blastocysts and requiring no breaching of the zona pellucida during the cleavage stage period of culture was presented. By using this method, interference with embryo development and extra stress due to long exposure to a suboptimal environment, as well as the potential dangerous effect of warming following laser shooting at the cleavage stage of development, can be avoided [Capalbo et al., 2014].

(4) PREPARATION OF CELLS FOR CGH, ACGH AND FISH analysis [in accordance with the information represented in the article written by Fragouli E., Alfarawati S., Daphnis D.D., Goodall N–n., Mania A., Griffiths T., Gordon A., Wells D. as the result of investigation of cytogenetic analysis of human blastocysts with the use of FISH, CGH and aCGH: scientific data and technical evaluation]

It is recommended by the experts that all TE biopsies should be washed in sterile phosphate–buffered saline solution with 0.1% polyvinyl alcohol, placed in microcentrifuge tubes and then stored at −80°C until they are processed [Fragouli et al., 2011]. The remainder of the embryos should be spread onto microscope slides, using either a modified version of the methanol/acetic acid fixation method (30 embryos) [Vellila et al., 2002] or the Tween–HCl fixation method (22 embryos) [Harper et al., 1994].

Fluorescent in situ hybridization

FISH analysis of the fixed blastocysts took place using two or three sequential hybridizations. The protocol used was described previously [Colls et al., 2007]. A microscope slide containing lymphocyte nuclei from a normal male should be also processed during each of the FISH rounds, allowing the efficiency of probe hybridization to be monitored. Chromosomes 13, 15, 16, 17, 18, 21, 22, X and Y should be examined along with any other chromosomes that had given an abnormal CGH and/or aCGH result [Fragouli et al., 2011].

Embryo classification and FISH scoring criteria

Previously published scoring criteria [Munné et al., 1998] should be employed during the analysis of the fixed embryos. Classification of embryos after CGH, aCGH and FISH results should be combined – took place according to criteria published by Delhanty et al. (1997).

Comparative genomic hybridization

The CGH protocol employed for the analysis of the blastocysts should be validated and described in detail [Wells et al., 1999; Fragouli et al., 2006]. Briefly, TE cells can be lysed using proteinase K. Genomic 46, XY DNA, extracted from blood can be diluted to a concentration ranging between 0.5 and 1 ng/ml and used as reference, with which results from the TE sample can be compared. Degenerate oligonucleotide–primed PCR can be employed for the whole genome amplification of the TE samples and also for the 46, XY reference DNA [Fragouli et al., 2011].

Microscopy, image analysis and interpretation

Metaphase spreads can be observed with the use of fluorescent microscope with a cooled charge–coupled device system, and filters for the fluorochromes use. Ten metaphases can be captured on average per hybridization. Analysis and interpretation of the captured images utilize Cytovision CGH software that converts fluorescent intensities into a red–green ratio for each chromosome. A normal chromosome copy number is indicated by a minimal fluctuation of the red–green ratio from 1:1. Chromosome loss (CL) is associated with a fluctuation of the ratio in favour of the red colouration (below 0.80), while chromosome gain (CG) is seen as deviation of the ratio towards the green colouration (above 1.20) [Fragouli et al., 2011].

Microarray CGH

The blastocysts can be tested using the 24Sure™ Cytochip. This technology screens all 24 chromosomes for both gain and loss with a unique BAC pooling strategy, which –together with specially designed software – enables robust results to be reported on the chromosome status of the single cell. Similar to metaphase comparative genomic hybridization (CGH), the protocol employed consisted of the following steps: cell lysis; whole genome amplification of TE samples; fluorescent labelling and hybridization of the TE and ‘reference DNA’ samples; post–hybridization washes; scanning and analysis of images [Fragouli et al., 2011].

Lysis and whole genome amplification of TE samples can be achieved using the SurePlex kit. The entirety of this procedure should be performed according to the manufacturer’s instructions. To determine the success of the amplification, 5 μl of the products can be analyzed on a 1% agarose gel. Amplified products can be seen as smears whose fragment size range between 100 and 1000 bp with a median size of ∼400 bp. The fluorescence labelling system should be used for the labelling of the amplified TE samples and also for labelling an available reference DNA. Test and reference DNA co–precipitation, their denaturation, array hybridization and the post-hybridization washes all should take place according to protocols provided by the manufacturer. The hybridization time should range between 3 hours and 16 hours [Fragouli et al., 2011].

Scanning and image analysis and interpretation

A laser scanner should be used to excite the hybridized fluorophores, and to read and store the resulting images of the hybridization. The MAPIX software should be used to control the scanning of the microarray slides. The resulting images should be stored in TIFF format file and should be analyzed by the BlueFuse Multi analysis software. Chromosome profiles should be examined for gain or loss with the use of a 3 × SD assessment. TE samples should be classified as normal or aneuploid according to this assessment [Fragouli et al., 2011].

Summarizing the results obtained during the comprehensive examination of blastocysts using three different molecular cytogenetic methods (FISH, CGH and aCGH) it was postulated that confirmed that mosaicism is a common phenomenon during the final stage of embryo preimplantation development [Fragouli et al., 2011].

Embryos which are characterized as being mosaic, usually can carry a single post–zygotic anomaly, or two or more such errors. The blastocysts are classified as being mosaic aneuploid if they consist of two or more cell lines carrying different chromosome abnormalities. The mosaic embryos can have chromosome abnormalities arising after fertilization as their sole abnormality, whereas others can display a combination of meiotic and post–zygotic errors.

It should also be noted that only few blastocysts can be found to have a chaotic chromosome arrangement, composed of multiple different aneuploid cell lines [Fragouli et al., 2011]. What is essential to mention is that the use of comprehensive methods such as comparative genome hybridization (CGH) and array comparative genome hybridization (aCGH), both able to screen the entire genome of cells revealed that almost all chromosomes participated in aneuploidy events of both meiotic and post–zygotic origin. Both methods comparative genome hybridization (CGH) and array comparative genome hybridization (aCGH) are able to detect a wide variety of chromosome abnormalities, which were in their vast majority confirmed by subsequent FISH analysis of multiple cells. Discrepancies between comparative genome hybridization (CGH) and array comparative genome hybridization (aCGH) are attributed to the presence of mosaicism. In both cases where this occurs, FISH reveals that the different results obtained are due to the presence of karyotypically distinct cell lines within the same embryo. To verify or to concern the sensitivity of comparative genome hybridization (CGH) and array comparative genome hybridization (aCGH), it is essential to examine control lymphocyte slides, allowing the efficiency of individual experiments to be established. Usually, probe panel efficiencies ranged between 95 and 98% [Fragouli et al., 2011]. As far as the sensitivity of comparative genome hybridization (CGH) and array comparative genome hybridization (aCGH) is concerned, it was estimated that both methods are able to reliably detect a chromosome abnormality provided it is present in ≥30% of the biopsied TE cells [Fragouli et al., 2008; Schoolcraft et al., 2010]. However, neither comparative genome hybridization (CGH) nor array comparative genome hybridization (aCGH) are capable of identifying ploidy changes (loss/gain of an entire set of chromosomes, e.g. triploidy). Only FISH analysis can identify carrying varying amounts of polyploid cells [Fragouli et al., 2011].

Detecting mosaicism in trophectoderm biopsies: current challenges and future possibilities

Chromosome mosaicism in day 3 aneuploid embryos that develop to morphologically normal blastocysts in vitro

Embryonic mosaicism, defined as the presence of karyotypically distinct cell lines within an embryo, has been frequently reported with a high incidence in preimplantation embryos derived from IVF cycles and is thought to be one of the major biological limitations for the routine application of preimplantation genetic screening for aneuploidies (PGD–A). The incidence of mosaicism in preimplantation embryos is in fact reported to be between 4 and 90%. However, these data are in excessive contrast with what is known from clinical pregnancies, where true fetal mosaicism is observed in less than 0.5% of cases. Therefore, these previous observations should be challenged in preimplantation embryos, presenting an alternative perspective, which also considers the impact of technical variation to diagnose mosaicism as one possible cause contributing to overestimation of the incidence of mosaicism in embryos. Although euploid/aneuploid mosaicism may be present in blastocysts, the possibility of detecting this phenomenon within a single trophectoderm biopsy represents a contemporary challenge to bring about improvement to the practice of preimplantation genetic screening for aneuploidies (PGD–A) [Capalbo et al., 2017].

Embryonic mosaicism is defined as the presence of karyotypically different cell lines within the same individual, such as a preimplantation embryo [Delhanty et al., 1997; Youssoufian and Pyeritz, 2002]. The primary origin of embryonic mosaicism is post–zygotic chromosome segregation errors as a consequence of mitotic non–disjunction. While other mechanisms have been considered, such as anaphase lag, and endoduplication or deletion, these events may be extremely rare [Gueye et al. 2014]. Although meiotic aneuploidies are uniformly present in all cells and present with a well–defined clinical penetrance in reproductive health [Hassold and Hunt, 2001; Cohen, 2002], the embryonic fate and the clinical consequences of mosaic aneuploidies may depend on many variables, including which chromosome is involved, when the error occurs and thus what percentage of the embryo is aneuploid, and where it is located within the embryo [Johnson et al., 1990; Wapner et al., 1992; Wilkins–Haug et al., 1995]. As a consequence, the clinical penetrance of a mosaic aneuploidy can be seen as unique for each event and difficult to be predicted in the absence of a well–defined phenotype.

Despite the fact that chromosomal mosaicism is diagnosed in <2% of prenatal specimens and only a small proportion of them (≈10%) is then confirmed in the fetus [Malvestiti et al., 2015], estimates of preimplantation stage mosaicism frequency range from 4% to as high as 90% [Taylor et al., 2014]. Indeed, these estimates are believed to be a major biological limitation to the success of preimplantation aneuploidy screening [Mastenbroek and Repping, 2014]. Moreover, the development of tools that might provide better sensitivity to the detection of low levels of aneuploidy in a mosaic trophectoderm (TE) biopsy has been proposed [Greco et al., 2015]. In turn, different laboratories have begun to report the diagnosis of mosaicism in clinical cases of blastocyst PGD for aneuploidies (PGD–A) [Capalbo et al., 2017]. As discussed before, mosaicism is expected to affect a minority of blastocysts, thus the development of comprehensive chromosome screening (CCS) technologies able to quantify the rate of abnormal cells in a mosaic euploid/aneuploid embryo is anticipated to provide an improvement in the clinical practice of PGD–A.

Comparative analysis of prevalence of chromosomal mosaicism in the preimplantation stage of embryo development: Cleavage stage versus Blastocyst stage in close scientific focus

Studies of mosaicism in cleavage stage embryos

One of the challenges facing preimplantation genetic screening for aneuploidies (PGD–A) is the developmentof accurate estimates of the frequency of chromosomal mosaicism in the preimplantation stage of development. Probably one of the most important limitations of prior estimates of embryonic mosaicism has to do withthe inaccuracy of single cell preimplantation genetic screening for aneuploidies (PGD–A) methods. In fact, all of the reported estimates of mosaicism are potentially impacted by the technical accuracy of methods used to predict aneuploidy. When analyzing multiple single cells from an embryo it is close to impossible to distinguish between technical artefacts, and a genuine biological variation due to mosaicism. This is especially relevant for cleavage stage embryos where multiple single cells from the same embryos are analyzed in separate reactions with a defined diagnostic error rates. Indeed, one single false positive aneuploidy observation is sufficient to result in a false positive diagnosis of mosaicism in the embryo. For example, suppose that a specific chromosome testing method is usedon single cells and has a 10% false positive error rate. The analysis of six normal blastomeres is expectedto result in a false positive mosaicism diagnosis in 50% of cases and 70% when 10 blastomeresare analyzed [Capalbo, et al., 2017]. It is not surprising then that FISH–based studies on single blastomeres have reported as high as 50–90% of cleavage stage embryos being mosaic since statistically the analysis of eight normal blastomeres with 15% false positive error rate is expected to result in a false positive mosaicism diagnosis rate of nearly 75% (probability of obtaining at least one event with a false positive discovery rate of 15%: 1–0.858). It is thus extremely crucial to take some rectification measures for this phenomenon and correct for the expected false positive rate of each method during the data analysis in order to obtain a more reliable estimation of the actual incidence of mosaicism [Capalbo, et al., 2017].

Another important consideration is the stringency of methods used to classify embryos as mosaic, which can vary significantly. For example, some studies only require one abnormal cell to be present in order to classify the embryo as mosaic. In contrast, the most rigorous criteria for classification would be the presence of reciprocal aneuploidy in two different cells or samples from the same embryo. That is, one biopsy displaying a monosomy of a specific chromosome and another biopsy from the same embryo displaying trisomy for the same chromosome.This level of evidence for a true mitotic non–disjunction event would minimize the impact of technical artefacts on estimates of mosaicism, and perhaps with a minimal reduction in the sensitivity of detection. It is consideredthat the degree of mosaicism is therefore likely to have been extremely overestimated due to the lack of correction for the expected false positives and to the non–standardized criteria used for classification of embryosas mosaic [Capalbo, et al., 2017].

More recently comprehensive chromosome screening (CCS) methods have been used to evaluate mosaicism in cleavage stage embryos [Wells and Delhanty 2000]. A high rate of mosaicism isn’t a unique phenomenon in cleavage–stage embryos [Munné et al., 1994; Delhanty and Handyside, 1995; Kuo et al., 1998; Vanneste et al., 2009a]. It is suggested that mosaic diploid/aneuploid embryos may result in chromosomally normal fetuses, as it was found the number of embryos that give rise to a successful pregnancy to be higher than the number of embryos that are normal diploid in every blastomere [Vanneste et al., 2009b]. Array comparative genomic hybridization (aCGH) was the most commonly used method for this purpose [Vanneste et al., 2009; Capalbo et al., 2013a, b; Mertzanidou et al., 2013a, b]. Even though advanced genetic technologies have been well adapted to work on single cells, they do not provide 100% accuracy and are still biased by amplification artefacts [Capalbo et al., 2015]. Depending on different procedural aspects, reagent batch and parameters for quality control and data analysis, a varying false positive error rate can be expected from the application of a specific array comparative genomic hybridization (aCGH) protocol on a single cell. Different versions of the analysis software and different protocols (dual channel array vs single channel array) may also impact performance [Capalbo, et al., 2017].

The introduction of higher resolution techniques such as the single–cell metaphase– and array–CGH has enabled the detection of de novo partial chromosome losses and gains. Chromosome breakage leading to chromosomal imbalance is reported in several studies using comparative genomic hybridization (CGH) [Wells et al., 1999; Voullaire et al., 2000, 2002; Wells and Delhanty, 2000]. Ideally, all studies investigating the presence of mosaicism with single cell analysis would first provide data on the methods accuracy on large dataset of single cells with previously established aneuploidies (positive controls). All array comparative genomic hybridization (aCGH) studies performed so far on blastomeres failed to report a primary validation on single cells, with the useof each specific protocol, to estimate the false positive error rate, or do not report such data on a sufficient numberof samples [Jacobs et al., 2014].

A good example is a recent publication of Mertzanidou A. and colleagues using array comparative genomic hybridization (aCGH) on all dissected blastomeres from 14 cleavage stage embryos. It was revealed and represented that around 70% of good–quality embryos carry chromosomal abnormalities, including structural aberrations. The main strength of that work compared with other published data is that: (I) the scientists analyzed the majority of the blastomeres of (II) top–quality embryos from a cohort of embryos with high implantation and developmental potential and for (III) all chromosomes. In that study, 20% of blastomere failed to produce a result due to amplification failure or low–profile quality and 70% of embryos were classified as mosaic [Mertzanidou et al., 2013b]. That study also provides an opportunity to indirectly evaluate the accuracy of detecting aneuploidy in general. It was postulated that high–level mosaicism and structural aberrations are not restricted to arrested or poorly developing embryos but are also common in good–quality in vitro fertilization (IVF) embryos [Mertzanidou et al., 2013b]. It is well established that the majority of aneuploidy is derived from maternal meiotic errors, at least one of the embryos in this study should display uniform aneuploidy for at least one chromosome. Surprisingly, no embryo contained the same aneuploidy in all of its cells. Furthermore, given that a substantial contribution to mosaicism derives from mitotic non–disjunction, it can be expected that at least one embryo should have displayed reciprocal errors for at least one chromosome [Daphnis et al., 2005; Munné et al., 2005; Mantikou et al., 2012; Capalbo et al., 2013b]. These previous observations in preimplantation embryos, presenting an alternative perspective, which also considers the impact of technical variation to diagnose mosaicism as one possible cause contributing to overestimation of the incidence of mosaicism in embryos were challenged by the experts Antonio Capalbo, Filippo Maria Ubaldi, Laura Rienzi, Richard Scott and Nathan Treff. They postulated that although euploid/aneuploid mosaicism may be present in blastocysts, the possibility of detecting this phenomenon within a single trophectoderm biopsy represents a contemporary challenge to bring about improvement to the practice of preimplantation genetic screening for aneuploidies (PGD–A). The purpose of this opinion paper is to provide a critical review of the literature, provide a possible alternative interpretation of the data, and discuss future challenges with diagnosing mosaicism in preimplantation genetic screening for aneuploidies (PGD–A) cycles.Theoretical and practical reanalysis of Mertzanidou A. concepts and their comparison with newer investigation’s concepts, represented in the article: “Detecting mosaicism in trophectoderm biopsies”, distinguished the controversial results: surprisingly, even though the high failure rate of analysis could have lowered the detection of non–disjunction, not a single reciprocal aneuploid chromosome was observed. While the authors suggested that mosaicism in embryos is predominant, this data can be alternatively interpreted as a display of the poor reliabilityof that specific array comparative genomic hybridization (aCGH) protocol and scoring criteria used when appliedto single cells [Capalbo, et al., 2017].

In contrast, another study involving characterization of mosaicism at the cleavage stage demonstrated a smaller prevalence by using a single nucleotide polymorphism (SNP) cell array based method of comprehensive chromosome screening (CCS) [Treff et al., 2010b]. Before applying the method to blastomeres, this single nucleotide polymorphism (SNP) array method was specifically evaluated for single cell accuracy using positive control cell lines and demonstrated 98.6% concordance with the expected karyotypes [Treff et al., 2010b]. In the analysis of mosaicism, blastomeres were randomized to either fluorescent in situ hybridization (FISH) or single nucleotide polymorphism (SNP) array analysis from arrested cleavage stage embryos. While fluorescent in situ hybridization (FISH) evaluated fewer chromosomes and fewer cells per embryo (because of lower reliability of obtaining a result compared to SNP array), it still estimated a rate of mosaicism of 100%. In contrast, single nucleotide polymorphism (SNP) array predicted a significantly lower mosaicism rate of only 31%, while also observing uniform aneuploidy as expected (meiotic errors). This study demonstrated that the use of inaccurate methods of aneuploidy screening can significantly overestimate the prevalence of mosaicism and further illustrates the caution necessary when interpreting results from poorly validated methods of preimplantation genetic screening for aneuploidies (PGD–A) [Treff et al., 2010b].

Furthermore, inaccurate predictions of mosaicism may also originate from differences in the cell cycle phase and deoxyribonucleic acid (DNA) replication stage of the blastomeres being studied. Current methodology does not take into account the phase of the cell cycle, despite the variable copy number (CN) status of different genomic regions in the S phase. The scientists suggest that in contrast, if one wants to study DNA replication in the cell, it is best to correct the log2 intensities ratios for the technical WGA GC bias (observed across G1– and G2/M–phase cells) and to further investigate these values using e.g. a moving average or a lenient PCF or CBS segmentation of the log 2 intensity ratio values. DNA replication is a fundamental process of life; however, many aspects of its modus operandi, certainly at a single–cell resolution, as well as its molecular links to other cellular processes remain elusive [Van der Aa et al., 2013]. It is known that the DNA of the S–phase cell is progressively replicated from multiple origins of replication and log2 ratios of single S–phase cells follow the patterns of early and late replication domains [Van der Aa et al., 2013; Dimitriadou et al., 2014]. Hence, at a given time point the genetic copy number (CN) profiling of an S–phase cell will demonstrate different loci with copy number (CN) status of 2, 3 or 4, depending on the replication status of this specific locus. This can possibly result in false positive and false negative copy number (CN) determinations if a sufficient portion of the chromosome is replicating and depending on the criteria used to define the copy number (CN) status. Hence, DNA imbalances may, on the one hand, be falsely interpreted as genuine aberrations in the S–phase cell’s copy number (CN) profile and hence lead to diagnostic error. This is especially relevant for cleavage stage embryos, where, fast dividing cells such as blastomeres or some tumour cells are expected to reside more often in S–phase, and thus the chances are higher to isolatea cell in S–phase from such a population. This is another potential source of error poorly acknowledged in existing studies, even thought no direct evidence has been reported so far, that whole chromosome aneuploidy call can be compromised by the cell cycle phase [Capalbo, et al., 2017]. Consequently, resuming the above–mentioned sources of false positive errors also provide a possible explanation for data reported regarding the generation of karyotypically normal embryonic stem cells from supposed aneuploid embryos after a fluorescent in situ hybridization (FISH) based PGD–A on single blastomeres as aneuploidy is commonly seenin preimplantation embryos, most particularly at the cleavage stage because of genome activation by third cell division [Bazrgar et al., 2013].

Studies of mosaicism in blastocysts

While many studies have investigated chromosomes in cleavage–stage embryos, the cytogenetics of blastocysts (5 or 6 days post–fertilization) has received comparatively little attention. With the growth in the use of blastocyst culture, there has been increasing interest in defining the types of chromosome errors persisting to the final stage of preimplantation development. Once again, FISH has been the main method employed for the examination of embryos at the blastocyst stage [Evsikov and Verlinsky, 1998; Magli et al., 2000; Sandalinas et al., 2001; Bielanska et al., 2002; Santos et al., 2010]. All studies have confirmed that chromosome abnormalities and mosaicism remain common at the blastocyst stage, but suggest that aneuploidy rates are reduced compared with the cleavage stage [Evsikov and Verlinsky, 1998; Coonen et al., 2004; Santos et al., 2010].

Recently, there have been efforts to provide a more detailed characterization of blastocyst cytogenetics, using methods such as comparative genomic hybridization (CGH) and single nucleotide polymorphism (SNP) microarrays [Fragouli et al., 2008; Northrop et al., 2010]. A detailed evaluation of all the chromosomes in 158 good–quality blastocysts revealed that even the most severe chromosomal abnormalities (e.g. monosomy, imbalance affecting the largest chromosomes and aneuploidy affecting multiple chromosomes) were often capable of surviving up to the blastocyst stage [Fragouli et al., 2008].

From the clinical perspective, there has been increasing interest in screening blastocyst-stage embryos for chromosomal abnormalities, with a view to detecting and preferentially transferring euploid normal embryos during IVF cycles. Early clinical data suggest that an approach combining blastocyst biopsy and comprehensive chromosome screening using comparative genomic hybridization (CGH) or microarray–CGH (aCGH)may represent the optimal approach for preimplantation genetic screening (PGS) [Fragouli et al., 2010; Schoolcraft et al., 2010]. However, there is little information concerning the accuracy of comparative genomic hybridization (CGH) applied to blastocyst biopsies and no published data validating aCGH on blastocyst biopsies. In particular, the impact of mosaicism on testing at the blastocyst stage remains unclear.

Scientific studies of mosaicism in blastocysts have reported much lower levels of compared to the cleavage stage. This observation has commonly been interpreted as a selection against embryos with mitotic derived aneuploidy between the cleavage and blastocyst stage of development. According to this model, it is indeed possible that blastomeres affected by a mitotic chromosome segregation error might harbour additional cellular defects causing a developmental arrest [Capalbo, et al., 2017]. However, since all types of uniform aneuploidies can survive to the blastocyst stage [Fragouli et al., 2011; Franasiak et al., 2014], including complex aneuploidies, an alternative explanation for the observed difference between cleavage and blastocyst stage mosaicism rate can be found in the improved accuracy achieved when evaluating multiple cells instead of single cells. This includes the fact that S–phase artefacts are less likely to impact predictions from a Trophectoderm (TE) biopsy. Of course, the reduced rate of mosaicism may also be due to masking of aneuploidy by euploid cells or by cells with the reciprocal aneuploidy within the same biopsy, resulting in overestimation of euploidy [Capalbo, et al., 2017].

The ideal approach to investigate the incidence and prevalence of mosaicism in blastocysts would entail dissection of the entire embryo into single cells and the use of a robust comprehensive chromosome screening (CCS) platform with a very low and well–defined error rate. However, this approach has been unattainable as a result of absence of an effective method to disaggregate blastocysts down to single cells. Alternative approaches to estimate mosaicism in blastocysts are single cell analysis by fluorescent in situ hybridization (FISH) or by comprehensive chromosome screening (CCS) analysis of multiple blinded biopsies [Capalbo, et al., 2017].

The scientists have reported an extensive study on chromosome mosaicism at the blastocyst stage [Capalbo et al., 2014] using FISH to analyze three Trophectoderm (TE) sections and the inner cell mass (ICM) of 70 blastocysts. In this study, inner cell mass (ICM) isolation methodology using Trophectoderm (TE) specific biomarkers was also validated. Furthermore, a mosaicism classification was made only if >10% of the nuclei presented with the same abnormality and if the same aneuploid signal was present in at least two cells from the same embryonic section. This criterion is currently one of the more straightforward approaches in cytogenetics to distinguish true aneuploidy from fluorescent in situ hybridization (FISH) artefacts due to false positive results and mosaicism diagnosis, when multiple nuclei are tested together and control material is lacking [Capalbo, et al., 2017]. What is essential to mention is Vysis’ multicentre study of FISH using AneuVysion applied to nuclei from uncultured amniocytes (Vysis, Inc., Downers Grove, IL, USA, 1997) indicated that a non–mosaic disomic result had >90% of nuclei with two signals and can be expected to have up to 10% of nuclei with deviant patter (i.e. not two signals, mostly one or three signals and skewed towards the former). Since in the analysis of few nuclei per sample, for instance eight, only one deviant pattern is required to surpass the 10% threshold, more than one deviant pattern was also included as criteria to provide evidence of mosaicism. That is the reason why a 10% threshold and the observation of at least two consistent aneuploid results in the analysis of the whole dissected blastocyst was used, trying to maximize the detection of genuine mosaicism cases, even though some low–grade mosaicism cases would be missed. Using these criteria of analysis, mosaic chromosomal errors were observed in 15.7% of the blastocysts tested, but only two cases (2.9%) embryos showed a mix of normal and abnormal cells (mosaic diploid/aneuploid). Of note, the scientists observed that the proportion of abnormal cells within the blastocyst predicted their distribution, i.e. embryos showing higher mosaicism rates had abnormal cells present across all blastocyst sections. Accordingly, high grade mosaicism cases that are expected to be of clinical relevance are expected to show abnormal cells in all blastocysts area, thus increasing the likelihood of detection in Trophectoderm (TE) based preimplantation genetic screening for aneuploidies (PGD–A) [Capalbo, et al., 2017]. This was confirmed in the study by comparing the original comprehensive chromosome screening (CCS) diagnosis obtained from clinical Trophectoderm (TE) biopsies with the actual chromosomal constitution of the dissected blastocysts [Capalbo et al., 2014]. Furthermore, no evidence of preferential segregation of aneuploidy in the inner cell mass (ICM) or Trophectoderm (TE) was observed. It has to be underlined that, as for all FISH–based studies, the analysis of nine chromosomes might have lowered the sensitivity of detection.

To summarize, considering that in prenatal specimens chromosomal mosaicism is detected in ~1–2%, that mosaicism associated with ART persists beyond the preimplantation embryo at a rate similar to that associated with pregnancies conceived spontaneously [Huang et al., 2009] and the possible methodological and technical flaws described above to assess its real incidence in preimplantation embryos, it is reasonable to speculate that chromosome mosaicism can be regarded as a relatively uncommon event in IVF embryos and should not have a major diagnostic impact on PGD–A cycles. The most extensive studies showed around 4–5% of blastocysts being mosaic diploid/aneuploid. Also, since there is no difference in the prevalence of mosaicism at the end of the first trimester in pregnancies conceived spontaneously compared with those with infertility treatments, the suggestion that inadequacies of embryo culture play a role in the genesis of this problem, increasing the risk of chromosome malsegregation during mitosis remains highly speculative at this time.

In any case, if mosaicism is reported, extensive genetic counselling should also be provided, including discussion of the technical limitations of defining the presence and extent of mosaicism from a single embryo biopsy, as well as the potential clinical consequences with respect to the actual chromosome involved. Clearly, utilization of methods that overestimate mosaicism will lead to inappropriate discard of healthy reproductively competent embryos [Capalbo et al., 2017].

One of the common misconceptions surrounding the ability of comprehensive chromosome screening (CCS) technology to detect mosaicism is that an intermediate alteration in the log2 ratio is definitive for making a prediction. Many experts have suggested that altered log2 ratios when observed in a Trophectoderm (TE) biopsy can be used to diagnose mosaicism [Greco et al., 2015; Munné et al., 2016]. Unfortunately, this criterion is insufficient as there are many possible alternative explanations for such observations, first and foremost being artifacts introduced by whole genome analysis (WGA) and aCGH or next generation sequencing (NGS) analyses on low input samples. There is a considerable risk of making an inaccurate prediction of mosaicism, resulting in a false positive diagnosis. This is a possible alternative explanation of the clinical cases reported in the recent report by Greco et al., (2015), where the authors suggest that embryos with mosaicism can implant and result in deliveryof normal babies. In fact, the authors fail to acknowledge an alternative explanation for the observations made,that the original diagnosis of mosaicism was incorrect and uniformly euploid were erroneously classifiedas mosaic [Capalbo et al., 2017].

The available data from investigation cell line mixture models can be disregarded on the rate of false positive mosaicism predictions by the experts in embryology. That is, sometimes they do not evaluate how often a chromosome is predicted mosaic when it should not have been. Furthermore, the experts should validate the ability to predict mosaicism by analyzing additional biopsies from the same embryo to establish predictive value. A more rigorous method for finding mosaicism in an embryo would be to identify reciprocal aneuploidy of the same chromosome in multiple biopsies (for instance, finding some cells with monosomy and other cells with trisomy of the same chromosome). Without appropriate clinical evaluation of the accuracy of using log2 ratios to predict mosaicism, it may be prudent to modify the interpretation of such observations to indicate that the pattern is consistent with possible mosaicism, rather than stating that the embryo is indeed mosaic. Furthermore, it should be important to evaluate reliability of each method to detect mosaicism in Trophectoderm (TE) biopsies for every single chromosome, as each one can have its own and different performance in the analysis [Capalbo et al., 2017].

CONCLUSION

It was proved that a combination of comprehensive cytogenetic screening and follow–up assessment of multiple cells can provide a unique insight into the chromosomes of embryos at the final stage of human preimplantation development. The use of comprehensive methods of chromosome screening (CGH, aCGH and FISH) provides a more complete picture of the extent and variety of chromosome abnormalities persisting to the blastocyst stage than has previously been possible, while FISH allowed large numbers of cells to be independently evaluated, shedding light on the extent of chromosomal mosaicism. Both CGH and aCGH performed equally well, FISH analysis of additional cells usually demonstrates good concordance between all three methods. If CGH and FISH agree for 94% of the examined embryos, and aCGH and FISH display 100% concordance. There are two discrepancies observed between CGH and aCGH, both of which are attributed to mosaicism [Fragouli et al., 2011]. This is confirmed by FISH analysis, which reveals that each of the cell lines detected by CGH and aCGH are present within the embryos. The reliability of aCGH in detecting chromosome abnormalities, even at the single cell level, has also been demonstrated in a recent study carried out by Gutierrez–Mateo et al. (2010).

The combination of comprehensive chromosome screening and follow–up with FISH permits the origin of each chromosome abnormality (meiotic or post–zygotic) to be estimated. Aneuploidies present in every cell of the embryo are presumed to be meiotic in origin, while those existing in mosaic form are considered mitotic.In addition, the blastocysts carrying meiotic anomalies, are found to have varying degrees of mosaicism.The blastocysts which display aneuploidies in every cell are classified as mosaic aneuploid or chaotic.The mosaic blastocysts which consist of a mixture of diploid and aneuploid cell lines are classified as mosaic diploid–aneuploid [Fragouli et al., 2011].

Findings obtained with the use of comprehensive chromosome screening methods such as CGH and SNP microarrays have shown that any chromosome can be affected by aneuploidy in blastocysts, and that many monosomies as well as trisomies survive to the final stage of human preimplantation development [Fragouli et al., 2008, 2010; Alfarawati et al., 2010; Northrop et al., 2010].

Previous cytogenetic studies examining blastocyst stage embryos have either assessed a restricted set of chromosomes in multiple cells (using FISH), or screened the entire set of chromosomes, but only in a small number of cells (via CGH) [Coonen et al., 2004; Daphnis et al., 2005, 2008; Fragouli et al., 2008, 2010; Santos et al., 2010]. Findings obtained with the use of comprehensive chromosome screening methods such as CGH and SNP microarrays have shown that any chromosome can be affected by aneuploidy in blastocysts, and that many monosomies as well as trisomies survive to the final stage of human preimplantation development [Fragouli et al., 2008, 2010; Alfarawati et al., 2010; Northrop et al., 2010]. In the current investigation represented by Fragouli E., Alfarawati S., Daphnis D.D., Goodall N–n., Mania A., Griffiths T., Gordon A. and Wells D., a combination of comprehensive cytogenetic screening and follow–up assessment of multiple cells provided a unique insight into the chromosomes of embryos at the final stage of embryo preimplantation development. Additionally, their study shed light on the reliability of a new generation of embryoscreening techniques. Such data are urgently required, as these methods are already being widely applied to assist identification of viable embryos (i.e. for the purpose of PGS) [Fragouli E., 2011]. The reliability of array comparative genomic hybridization (aCGH) in detecting chromosome abnormalities, even at the single cell level, has been demonstrated in a recent study carried out by Gutierrez–Mateo C. et al. (2010) [Gutierrez–Mateo C. et al., 2010] and Fragouli E. et al. (2011) [Fragouli et al., 2011]. Both CGH and aCGH were performed equally well and furthermore, the results were statistically evaluated for almost all of the examined samples. Specifically, the success rate for comparative genomic hybridization (CGH) was 98% (results for 51/52 TE samples) and for array comparative genomic hybridization (aCGH) it was 100% (20/20 samples) [Fragouli et al., 2011]. The success rates obtained during this investigation were similar to those of previously published studies that used comparative genomic hybridization (CGH) to examine TE samples [Fragouli et al., 2008, 2010] and are comparable to the success rates achieved using SNP microarrays reported in a recent study [Northrop et al., 2010]. The combination of comprehensive chromosome screening and follow–up with FISH permit the origin of each chromosome abnormality (meiotic or post–zygotic) to be estimated. Aneuploidies present in every cell of the embryo are presumed to be meiotic in origin, while those existing in mosaic form are considered mitotic.

Usually, the blastocysts carrying meiotic anomalies, are found to have varying degrees of mosaicism. The blastocysts of the mosaic embryos display aneuploidies in every cell and are classified as mosaic aneuploid or chaotic. The blastocysts consist of a mixture of diploid and aneuploid cell lines, and are therefore classified as mosaic diploid–aneuploid [Fragouli et al., 2011]. The few FISH–based studies of blastocysts previously undertaken had also suggested that mosaicism is common at this stage. Similarly, the results obtained with three–colour FISH analysis during the studies of Coonen E. et al. (2004), Daphnis D.D. et al. (2005) and Bielanska M. et al. (2005) demonstrated that mosaicism was present in more than half of all the embryos assessed.

Findings obtained with the use of comprehensive chromosome screening methods such as comparative genomic hybridization (CGH) and SNP microarrays have shown that any chromosome can be affected by aneuploidy in blastocysts, and that many monosomies as well as trisomies survive to the final stage of human preimplantation development [Fragouli et al., 2008, 2010; Alfarawati et al., 2010; Northrop et al., 2010].

There have been concerns that blastocyst analysis using comparative genomic hybridization (CGH), array comparative genomic hybridization (aCGH) or other microarray–based methods might fail to provide an appropriate diagnosis for mosaic embryos. However, in the Fragouli et al. (2011)study, half of the mosaic embryos contained no normal cells, and would therefore have been correctly classified as ‘abnormal’ regardless of which cell was biopsied. Of more significance may be the 17% of blastocysts that contained both diploid and aneuploid cells. The fate of these embryos is unclear. It is possible that the aneuploid cells have a growth disadvantage or are eliminated by processes such as apoptosis, leading to a decline in their numbers as development progresses, ultimately resulting in a normal fetus. If this scenario is correct, some diploid–aneuploid mosaic blastocysts may be viable and might be incorrectly discarded if aneuploid cells were detected following TE biopsy. However, cell number is an important aspect of embryo potential, and it therefore seems unlikely that embryos with a low proportion of normal cells would have much capacity for further development. Mosaic diploid–aneuploid blastocysts in which at least 50% of the cells were chromosomally normal accounted for <6% of blastocysts analyzed [Fragouli et al., 2011]. It is also worth noting that there is no evidence for preferential allocation of abnormal cells to the TE in preimplantation embryos. Studies suggest that TE samples provide an accurate indication of the chromosome constitution of the inner cell mass in the vast majority of cases [Evsikov and Verlinsky, 1998; Magli et al., 2000; Fragouli et al., 2008; Northrop et al., 2010].

To conclude, Fragouli E. et al. (2011) is the first study to combine comparative genomic hybridization (CGH), array comparative genomic hybridization (aCGH) and FISH analysis of preimplantation embryos. The results obtained by the scientists Fragouli E. et al. (2011) provide validation of the new comparative genomic hybridization (CGH) and array comparative genomic hybridization (aCGH) embryo screening methods and also yield the most detailed information to date on the rate of chromosome abnormalities and mosaicism at the blastocyst stage [Fragouli et al., 2011]. The results indicate that almost half of the investigated blastocysts contained no euploid cells, in most cases due to the presence of a meiotic error. Mosaicism was also common, affecting a third of the embryos tested. However, many mosaics contained no normal cells. It is suggested that embryo screening at the blastocyst stage using comparative genomic hybridization (CGH) or array comparative genomic hybridization (aCGH) is likely to be an accurate tool for detecting chromosomally normal blastocysts and may assist in identifying viable euploid embryos with higher implantation potential [Fragouli et al., 2011].

Biopsy is now either at the polar body or at the blastocyst stage of development, following the assumption that this is less detrimental to the embryo and that this avoids the negative effect of mosaicism on efficacy. Ploidy status is now determined using comparative genomic hybridization (CGH) arrays and single nucleotide polymorphism (SNP) arrays, allowing the analysis of all chromosomes with proclaimed greater accuracy than FISH [Wells et al., 2008].

There is evidence that embryos that are diploid–aneuploid mosaic at the cleavage stage but are still developing with a normal cleavage rate and pattern can reach the blastocyst stage [Gonzalez–Merino et al., 2003; Baart et al., 2006; Vanneste et al., 2011], although this does not imply that they all would implant. It has been suggested that embryos might ‘self–correct’ their chromosome complement as they develop towards the blastocyst stage [Rubio et al., 2007; Barbash–Hazan et al., 2009; Vanneste et al., 2009; Robberecht et al., 2010]. Several studies show that the proportion of aneuploid cells in embryos diminish as the embryos go through the cleavage, morula and blastocyst stage [Bielanska et al., 2002; Gonzalez–Merino et al., 2003]. Several mechanisms have been suggested to explain the ‘self–correction’, such as preferential allocation of diploid cells to the inner cell mass, loss of aneuploid cells due to apoptosis or trisomic rescue by anaphase lagging or non–disjunction [Kalousek, 2000; Los et al., 2004; Robberecht et al., 2010]. Although our data do not shed light on this question, they represent a reference set for further scientific investigations.

Of additional concern is the potential harm to the embryo caused by the biopsy procedure, as this harm cannot yet be fully excluded [De Vos and Steirteghem, 2001; Scott et al., 2013b]. Also, the exact prevalence of mosaicism at the blastocyst stage using the new methods for analysis is yet unknown, and with that a potential detrimental effect of mosaicism on Preimplantation Genetic Diagnosis (PGS) effectiveness cannot be fully excluded [van Echten–Arends et al., 2011]. Large studies on re–analysis of embryos after Preimplantation Genetic Diagnosis (PGS) using the new methods have still to be published. And for most new methods, diagnostic accuracy has yet to be determined. Preimplantation Genetic Diagnosis (PGS) is a black and white test that divides embryos into those that can be transferred and those that should be discarded. When this is done with <100% accuracy, either due to technical failure or mosaicism, potentially viable embryos are discarded and this will potentially even lower the pregnancy rate after IVF. For Preimplantation Genetic Diagnosis (PGS) using FISH, this appeared to be one of the problems [Scriven and Bossuyt, 2010]. For the new methods of analysis, which are generally assumed to be more accurate, surprisingly limited data on diagnostic accuracy is available considering the fact that they are already being used clinically. For most methods, it still has to be determined what the sensitivity (correctly identified embryos), specificity (correctly identified diploid embryos), positive predictive value (proportion of aneuploid embryos) and negative predictive value (proportion of diploid embryos) actually are.

These doubts make high–quality evidence even more needed before routine clinical application of Preimplantation Genetic Diagnosis (PGS). Preimplantation Genetic Diagnosis (PGS) should still only be offered by means of rigorously designed, conducted and reported randomized trials, if at all. Whereas Preimplantation Genetic Diagnosis (PGS), or any other embryo selection technique could indeed select better embryos, resulting in improved implantation rates or pregnancy rates per transfer, it could at the same time result in less transfers or less embryos to transfer by selecting out embryos, resulting in lower pregnancy rates per woman or per started cycle.

Nowadays it is widely recognized, even by the firmest criticasters of the first trials, that Preimplantation Genetic Diagnosis (PGS) as it had been applied for over a decade, with FISH analysis of blastomeres aspirated on Day 3 of embryo development, was of no use [Brown, 2014]. The mosaic nature of preimplantation embryos at this developmental stage, i.e. the observation that not all cells have the same chromosomal constitution, and the technique used, i.e. FISH analysis of a limited number of chromosomes with limited accuracy, are considered the main reasons for the inefficacy of PGS [Scriven and Bossuyt, 2010; van Echten–Arends et al., 2011].

Mosaicism has been likely overestimated by the imperfect nature and lack of robustness of methods of testing and it does not seem to be increased in IVF cycles compared to natural conceptions. Accordingly, chromosome mosaicism is not expected to be a major biological limitation for the systematic application of PGD–A in IVF. Methods of detecting aneuploidy within a mosaic Trophectoderm (TE) biopsy are under development but may only provide marginal improvements to the clinical application of PGD–A due to the expected low incidence of this biological phenomenon in blastocysts and pregnancies and the fact that there will always exist a sampling error when estimating the presence of mosaicism from a single biopsy. The use of altered log2 ratio data to predict mosaicism must be considered with criticism in order to avoid erroneous discard of embryos with reproductive potential. When results indicate the potential for mosaicism to exist, it is inappropriate to report a genuine diagnosis of mosaicism but it should instead be classified as a pattern ‘consistent with possible mosaicism’ and be accompanied by extensive genetic counselling which recognizes the many limitations of such a prediction. Future works should also focus on developing level I evidence of the clinical predictive value of new mosaicism classification schemes [Capalbo et al., 2017].

In conclusion, we would like to represent evidence that around 70% of good–quality embryos carry chromosomal abnormalities, including structural aberrations. According to statistical analysis data, presented in study “Microarray analysis reveals abnormal chromosomal complements in over 70% of 14 normally developing human embryos”, the inclusive structure of transparent and integrative analysis of embryo’s abnormal chromosomal complements was revealed: (I) it should be analyzed the majority of the blastomeres of (II) top–quality embryos from a cohort of embryos with high implantation and developmental potential and (III) all chromosomes [Mertzanidou et al., 2013]. These experiments should elucidate the true frequency and biological significance of chromosomal instability and the natural course of aneuploid cells in a normally developing embryo.